Abstract 3725: <i>SETD2</i> loss in renal carcinoma cells induces the unfolded protein response
Authors: Alexander Metz, Marya Kozinova, Robert Uzzo, Jessica Peskin, Michael Slifker, Janusz Franco-Barraza, Edna Cukierman, Philip Abbosh
Journal: Cancer Research (2022)

Abstract Introduction: SETD2 encodes a histone H3-K36 methyltransferase which is frequently inactivated in clear cell renal carcinomas (ccRCCs) and papillary RCCs via 3p deletion/LOH and deleterious mutations. Histone H3-K36 trimethylation is facilitated by SETD2, which is necessary for proper pre-mRNA intron splicing. Improperly spliced mature mRNA may lead to aberrant translation of retained introns (ATaRI), which represents potential therapeutic vulnerabilities. We explored this hypothesis using real world data and RCC models. Methods and Results: Gene set enrichment analysis comparing SETD2-mutant to WT tumors using samples from the TCGA KIRC data set revealed that the unfolded protein response (UPR) was strongly enriched, as well as several immunotherapy-relevant pathways. This suggested that peptides arising from ATaRI may be present, since they would not be expected to fold properly and thus need to be addressed by the UPR pathway to maintain homeostasis. To investigate this further, we generated Setd2-isogenic RENCA cells using CRISPR. Knockout was confirmed by sequencing and immunoblot. H3K36 trimethylation was decreased or eliminated in monoclonal knockout cell lines, confirming a functional effect. Markers of UPR activation, including Atf4 and cleaved Atf6, were found to be upregulated in Setd2-mutant RENCA cells compared to controls as measured by immunoblot. Cleaved ATF6 translocates to the nucleus to induce the UPR transcriptional program. Consistent with this, Atf6 was found to localize to the nucleus in Setd2-knockout cells using immunofluorescence (IF). Analysis of tissue microarrays of human SETD2-mutant vs -WT ccRCC revealed increased ATF6 signal in areas with low H3K36 tri-methylation, indicating UPR activation in vivo. Interestingly, CHOP, another downstream effector of the UPR pathway which predominantly regulates cell death, did not become upregulated in Setd2-knockout cells, suggesting activation of a compensatory cell survival pathway. Geldanamycin was shown to destabilize the Perk and Ire1a arms of the UPR, which resulted in increased cell death in Setd2 mutant cells using Cell Titre glo. Additionally, H3K36 trimethylation has been implicated in directing homologous repair of DNA, and blunted responses to agents that induce DNA double-strand breaks of p-Atm, p53, and Rad51 were observed by immunoblot and IF. The inability to detect DNA damage in Setd2 mutant cells however did not confer sensitivity to PARP inhibitors. Conclusions: We identify activation of the UPR upon Setd2 loss and suggest that activation of part of the UPR pathway may represent a new therapeutic vulnerability for exploitation as a rationale for personalized medicine. We further characterize the altered DNA damage response in the setting of Setd2 loss. We continue to evaluate the generation of peptides arising from ATaRI in Setd2-mutant contexts. Citation Format: Alexander Metz, Marya Kozinova, Robert Uzzo, Jessica Peskin, Michael Slifker, Janusz Franco-Barraza, Edna Cukierman, Philip Abbosh. SETD2 loss in renal carcinoma cells induces the unfolded protein response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3725.